Scripps Scientists Describe Drug-Resistance Protein

Newswise — Scientists at The Scripps Research Institute have solved the structure of a protein called MsbA that is involved in resisting antibiotics and chemotherapy. The structure of this membrane transporter protein is described in the current issue of the journal Science. Bacteria use these transporters to nullify antibiotics, and human cancer cells have similar membrane transporters on their surfaces that undermine the potency of chemotherapy drugs. "We actually have very good drugs to fight cancer and to kill bacteria," says Assistant Professor Geoffrey Chang, Ph.D., of the Department of Molecular Biology. "[But] they can't always get in the cells to work." The structures Chang and Research Associate Christopher Reyes solved using high-resolution x-ray crystallography reveal molecular details that could be useful for improving cancer therapy and fighting antibiotic-resistant bacteria. Wonder Drugs and Super Bugs At the dawn of the 20th century, bacterial infections accounted for several of the leading causes of death in the United States. Then came the antibiotic revolution. Antibiotic "wonder drugs" toppled tuberculosis (TB) and typhoid fever, controlled cholera and gonorrhea, reduced staphylococcal dysentery, and lowered the incidence of many other pandemic bacterial infections. These antibiotics are basically natural chemicals (or derivatives of natural chemicals) produced by other bacteria or fungi in the environment to kill off the competition. Scientists in the last century have discovered a number of these natural "antibiotic" products that have been used as the basis for treating bacterial infections. By the middle of the century, the threat posed by many types of bacteria seemed to be waning. Bacterial infections that once topped the list as leading causes of death in the United States were no longer among the top ten. The average life expectancy in the United States soared from 47.3 years in 1900 to almost 80 years today, and antibiotics are partly to thank for this. But in the last few decades, mutant strains of several types of bacteria with the ability to resist antibiotics have emerged, including those that cause TB, pneumonia, cholera, typhoid, salmonella, and staphylococcal dysentery. The bacteria that were once contained by drugs are now outstripping the ability of drugs to contain them, and as a result, says Chang, "What's happening today is that a lot of these diseases are coming back." These diseases are coming back resistant to the antibiotics that have been used to treat them, and people infected with these resistant strains must be treated with alternative antibiotics. Now some super bugs -- multiple drug-resistant bacteria -- are emerging as an even greater threat. Multiple drug-resistant TB is no longer susceptible to broad categories of antibiotics, such as rifampicin, isoniazid, and streptomycin. Some strains of the common hospital infection-causing bacteria Staphylococcus aureus are resistant to all antibiotics except vancomycin, which is a drug of last resort, and some strains of Streptococcus pneumoniae are even resistant to vancomycin. Certain strains of Shigella dysenteriae, the cause of epidemic dysentery, have even become resistant to all but a single drug -- the quinolone ciprofloxacin -- and may soon become completely untreatable. This is a major concern for public health because, according to the World Health Organization (WHO), large-scale epidemics of dysentery driven by this pathogen have been known to cause tens of thousands of deaths in Central America, South Asia, and central and southern Africa. Treating multiple drug-resistant bacterial infections can be a hundred times more expensive than treating normal infections, and the WHO estimates the total cost of treating all hospital-borne antibiotic resistant bacterial infections is around $10 billion a year. Worse, with modern rapid transit and world travel, multiple drug-resistant bacteria could potentially spread beyond the isolated confines of a hospital and into the general population. Resistance to Antibiotics and Chemotherapy Bacteria resist antibiotic drugs in a number of ways. Classical antibiotics target essential machinery in bacterial cells, such as protein synthesis, nucleic acid replication, and cell wall synthesis. Bacteria acquire antibiotic resistance by doing things like encoding enzymes that degrade antibiotics or sequester antibiotics by binding to them, undergoing small point mutations in the molecular targets that lower a drug's affinity, and overproducing a drug's substrate in the cell. However, another way that bacteria resist antibiotic drugs is by using membrane transporters -- large proteins that sit in the cell membrane and move other molecules in and out of the cell. One of these transporters, the structure of which is the subject of Chang and Reyes' recent Science paper, is called MsbA. It belongs to the ATP Binding Cassette (ABC) transporter molecule family. ABC transporters are ubiquitous on the cell surfaces of almost all organisms. This is one of the largest superfamilies of transporter molecules. They transfer drugs, sugars, and peptides in organisms from bacteria to humans. MsbA molecules play an essential role for bacteria because they help them build their cell walls by flipping molecules like "lipopolysaccharide" (LPS) and "lipid A" from the inner membrane to the outer membrane. These molecules are components of bacterial cell walls, and flipping them from the inner to the outer membrane of bacteria is necessary for bacterial cell growth. The structure is important for a number of reasons. One is that solving the structure helped Chang and Reyes to propose a mechanism by which it works. The structure of MsbA is a dimer with two identical subunits. These subunits stretch across the cell membrane, coming together at the top (outside of the cell) and opening up like two outstretched arms on the inside of the cell.

Significantly, the structure is trapped in what they call a "post-hydrolysis" state -- basically caught in the act of flipping an LPS molecule. "You get to see the molecule half-cocked in action, just before the lipid is flipped outside," says Chang. When the arms encounter LPS or lipid A, they close around the polar part of the amphipathic molecule, flip it over, and send it through the top to the other side of the membrane. This is most likely the same thing that happens when other drug pumps transport antibiotics out of the cell, and the structure of MsbA may help scientists design compounds to block its action. Coming up with a way to block this transporter would potentially make antibiotics more potent. Any potential MsbA blocker might also have the dual effect of weakening the bacteria since many different bacteria use the same transporters for flipping LPS out of the cell and erecting a major barrier that blocks antibiotics from entering. Blocking MsbA could prevent this. "It's an Achilles heel," says Chang. "Without being able to flip LPS outside, a bacteria cannot build its outer membrane." The solved structure may also lead to ways of improving cancer chemotherapy. Humans have proteins called multidrug resistance transporters that are orthologous to MsbA (similar function and the same evolutionary ancestor). In human cells, these transporters play an essential protective role by removing harmful toxins, and transporter proteins are often found in the human gut, colon, and urinary tract. They are also found in mammary tissue, where they are involved in transporting lipids into milk ducts during lactation. Unfortunately, this protective role can reduce the efficacy of certain cancer treatments, says Chang, since the drugs are perceived as toxins. Having a high resolution structure such as the one Chang and Reyes solved could open the door for scientists to design a new class of drugs that patients would take in conjunction with antibiotic or chemotherapeutic agents to keep those drugs in the cells and increase their efficacy. Finally, this structure is significant because it belongs to a class of proteins -- the membrane proteins -- that have been among the most difficult structures to study because they are notoriously hard to solve. Less than one half of one percent of the structures contained in the Brookhaven National Laboratory Protein Data Bank are of integral membrane proteins, despite the fact that over a third of all proteins in the body are in the membrane. The research article, "Structure of the ABC Transporter MsbA in Complex with ADP Vanadate and Lipopolysaccharide" is authored by Christopher L. Reyes and Geoffrey Chang and appears in the May 13, 2005 issue of the journal Science. The research was funded in part by the National Institutes of Health, Beckman Young Investigators Grant, Fannie E. Rippel Foundation, the Baxter Foundation, and the Skaggs Institute for Research.

About the Scripps Research Institute and Scripps Florida The Scripps Research Institute in La Jolla, California, and Palm Beach County, Florida, is one of the world's largest, independent, non-profit biomedical research organizations. It stands at the forefront of basic biomedical science that seeks to comprehend the most fundamental processes of life. Scripps Research is internationally recognized for its research into immunology, molecular and cellular biology, chemistry, neurosciences, autoimmune, cardiovascular, and infectious diseases, and synthetic vaccine development. The Scripps Research Institute employs approximately 3,000 scientists, postdoctoral fellows, scientific and other technicians, doctoral degree graduate students, and administrative and technical support personnel in 14 buildings overlooking the Pacific Ocean in La Jolla, a part of the City of San Diego. Scripps Florida will be a 350,000 square-foot, state-of-the-art biomedical research facility to be built on 100 acres of undeveloped land in Palm Beach County. Scripps Florida will focus on basic biomedical science, drug discovery, and technology development, employing more than 500 researchers and support staff by 2010. Palm Beach County and the State of Florida have provided start-up economic incentives for development, building, staffing, and equipping the campus. Scripps Florida is now operating with more than 100 researchers and technicians at a 40,000 square-foot facility on the campus of Florida Atlantic University in Jupiter.